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Biomedical subjects

M Tonetti

Publications and source records attributed to M Tonetti.

At least 19 recordsLinked to original sources
PubMed2006

Supportive periodontal therapy using mechanical instrumentation or 2% minocycline gel: a 12 month randomized, controlled, single masked pilot study.

OBJECTIVE: To compare the short-term performance of subgingival local delivery of 2% minocycline gel and conventional subgingival debridement in supportive periodontal therapy (SPT) patients. METHODS: Forty adult patients having completed active treatment for moderate to advanced chronic periodontitis were included in a randomized, controlled, single masked maintenance care pilot study. Sites with residual pocket probing depths > or =5 mm and bleeding on probing were treated with either minocycline gel (minocycline-group) or scaling and root planing only (debridement-group) at baseline, 3, 6, and 9 months. Clinical and microbiological examinations were performed at baseline, 3, 6, 9, and 12 months. RESULTS: Full-mouth plaque and bleeding scores remained <10% and <20%, respectively, for both groups throughout the study. In both groups there was a persistent reduction in number of teeth and sites with probing pocket depths > or =5 mm (p<0.05) with no significant differences between the groups. The prevalence of Porphyromonas gingivalis, Tannerella forsythia, Treponema denticola, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Prevotella nigrescens, remained at levels < or =10(5) in the majority of patients and sites in both groups. CONCLUSION: This pilot study failed to show a difference between local delivery of 2% minocycline gel as mono-therapy and traditional subgingival debridement in patients on SPT.

Adult↗
PubMed2005

Functional gene polymorphisms in aggressive and chronic periodontitis.

There is strong evidence that genetic as well as environmental factors affect the development of periodontitis, and some suggestion that aggressive and chronic forms of the disease share the same genetic predisposition. This study addresses the hypothesis that there are both shared and unique genetic associations in these forms of periodontitis. A sample of 51 patients with aggressive disease, 57 patients with chronic disease, and 100 healthy controls was recruited for this study. Ten functional polymorphisms in 7 candidate genes were genotyped. The results show statistically significant (p <or= 0.05) differences between genotype frequencies in aggressive and controls (IL-1B +3954 & IL-6 -174); chronic and controls (IL-6 -174 & VDR -1056); chronic and aggressive periodontitis (IL-1A -889); and periodontitis as a whole and controls (VDR -1056, TLR-4 399 & IL-6 -174). These results suggest that there are in fact both shared and unique genetic associations in aggressive and chronic periodontitis.

Alleles↗
PubMed2004

Gene expression profiling of bone cells on smooth and rough titanium surfaces.

Titanium (Ti) and Ti alloys are widely used as dental and orthopaedic implants, but the effects of the surface characteristics of these materials on the response of cells and target tissues is not well understood. The present study has therefore examined the effects of a rough Ti (RT) and a smooth Ti (ST) surface on human bone cells in vitro. Scanning electron microscopy showed attachment and spreading of cells on both surfaces. Expression profiling using ATLAS gene arrays showed marked differences in gene responses after 3 h of culture. A number of osteoblast genes were identified as "roughness response" genes on the basis of changes in expression on the RT compared with the ST surfaces. The surface roughness of Ti was thus found to have a profound effect on the profile of genes expressed by the bone cells, and suggests that improvements in the biological activity and possibly the clinical efficacy of these materials could be achieved by selective regulation of gene expression mediated by controlled modification of Ti surface.

Adult↗
PubMed2004

Roughness response genes in osteoblasts.

Titanium (Ti) and Ti alloys are widely used as dental and orthopedic implants, but the effects of the surface characteristics of these materials, including roughness, on the response of target tissues in vivo are not well understood. The present study has therefore examined the effects of a moderately rough (sand-blasted, acid-etched; SLA) Ti surface, a highly rough (plasma-sprayed; TPS) surface, and a smooth surface (SMO) on bone cells in vitro. X-ray photoelectron spectroscopy showed that these surfaces had similar surface chemistry, while scanning electron microscopy suggested that the SLA provided a transiently less biocompatible surface, with initially less well-attached cells. SLA also delayed bone cell growth compared with SMO, whereas the TPS surface elicited the greatest increase in cell numbers. In addition, expression profiling using the ATLAS gene array showed marked differences in gene responses after 3 h of incubation; this increased further after 24 h, with TPS generating the largest number of up- and down-regulated genes compared with SLA and SMO. A number of osteoblast genes were also identified as 'roughness' genes on the basis of their similar response on SLA and TPS, compared with SMO. These findings show, for the first time, that the surface roughness of Ti has a profound effect on the profile of genes expressed by bone cells and suggest that improvements in the biological activity and possibly the clinical efficacy of these materials could be achieved by selective regulation of gene expression mediated via modification of surface roughness.

Adult↗
PubMed2002

Expression profiling of periodontal ligament cells stimulated with enamel matrix proteins in vitro: a model for tissue regeneration.

Several studies have examined the role of enamel matrix proteins in root formation and periodontal regeneration, although most of these have focused on a few specific genes which had previously been implicated. However, recent advances in expressional profiling have made it possible to examine the range of genetic responses involved in these processes. In the present experiments, we have therefore utilized this technique to determine the effects of enamel matrix proteins on the gene activities of periodontal ligament cells in vitro. Such cells were found to have an elevated level of RNA synthesis compared with control cells. Moreover, hybridization of the cDNA prepared from this RNA to gene array filters showed that there was differential expression of 121 genes, most of which had not previously been associated with periodontal regeneration. Some of these selective changes in gene activity might thus reflect the fundamental events that underlie periodontal development.

Adult↗
PubMed2002

Quality of RCTs in periodontology--a systematic review.

Randomization, concealment of treatment allocation, blinding, and patient follow-up are key quality components of randomized controlled trials (RCTs). The objective of this study was to assess the quality of RCTs in periodontology using these evidence-based components. Following a detailed search, screening and quality assessments of RCTs were conducted in duplicate and independently. The results showed that although 91% of trials were described as randomised, adequate methods for randomization and allocation concealment were found in 17% and 7% of studies, respectively. Blinding was adequate for the caregiver in 17% and for the examiner in 55% of studies. A clear accounting of all participants was present in 56% of reports. This rigorous systematic review revealed that the quality of RCTs in periodontology, judged by their publications, frequently does not meet recommended standards. If this quality is reflected in actual study conduct, fundamental errors could have a significant impact on the outcomes of these trials.

Dental Research↗
PubMed2001

Kinetic and crystallographic analyses support a sequential-ordered bi bi catalytic mechanism for Escherichia coli glucose-1-phosphate thymidylyltransferase.

Glucose-1-phosphate thymidylyltransferase is the first enzyme in the biosynthesis of dTDP-l-rhamnose, the precursor of l-rhamnose, an essential component of surface antigens, such as the O-lipopolysaccharide, mediating virulence and adhesion to host tissues in many microorganisms. The enzyme catalyses the formation of dTDP-glucose, from dTTP and glucose 1-phosphate, as well as its pyrophosphorolysis. To shed more light on the catalytic properties of glucose-1-phosphate thymidylyltransferase from Escherichia coli, specifically distinguishing between ping pong and sequential ordered bi bi reaction mechanisms, the enzyme kinetic properties have been analysed in the presence of different substrates and inhibitors. Moreover, three different complexes of glucose-1-phosphate thymidylyltransferase (co-crystallized with dTDP, with dTMP and glucose-1-phosphate, with d-thymidine and glucose-1-phosphate) have been analysed by X-ray crystallography, in the 1.9-2.3 A resolution range (R-factors of 17.3-17.5 %). The homotetrameric enzyme shows strongly conserved substrate/inhibitor binding modes in a surface cavity next to the topological switch-point of a quasi-Rossmann fold. Inspection of the subunit tertiary structure reveals relationships to other enzymes involved in the biosynthesis of nucleotide-sugars, including distant proteins such as the molybdenum cofactor biosynthesis protein MobA. The precise location of the substrate relative to putative reactive residues in the catalytic center suggests that, in keeping with the results of the kinetic measurements, both catalysed reactions, i.e. dTDP-glucose biosynthesis and pyrophosphorolysis, follow a sequential ordered bi bi catalytic mechanism.

Amino Acid Sequence↗
PubMed2001

Impairment of the Golgi GDP-L-fucose transport and unresponsiveness to fucose replacement therapy in LAD II patients.

Leukocyte adhesion deficiency type II is an autosomal recessive syndrome characterized by generalized reduction of L-fucose in glycoconjugates; the specific molecular defect is still undefined. The most important clinical symptoms include severe growth and mental retardation and severe immunodeficiency. Patients from two ethnic groups have been reported, i.e. Arab and Turkish. We have observed that GDP-L-fucose transport into Golgi vesicles was specifically impaired in an Arab patient, with a significant reduction of the V:(max) but no significant differences in the K:(m) from control and parents. GDP-L-fucose transport showed simple saturation kinetics in all samples. Transport of UDP-galactose, UDP-N:-acetylglucosamine, and CMP-sialic acid was comparable into vesicles from the Arab patient, parents, and control. These kinetic parameters probably account for the failure to obtain any clinical and biochemical response to fucose therapy in Arab patients. This contrasts both with the distinctive kinetic properties of GDP-L-fucose transport and with the success of fucose therapy, which have been recently reported in one patient of Turkish origin. Accordingly, the biochemical properties of GDP-L-fucose transport into the Golgi are consistent with different variants of leukocyte adhesion deficiency type II that are probably the result of different molecular defects.

Biological Transport↗
PubMed2000

Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose Epimerase/Reductase by kinetic and crystallographic characterization of site-specific mutants.

GDP-4-keto-6-deoxy-d-mannose epimerase/reductase is a bifunctional enzyme responsible for the last step in the biosynthesis of GDP-l-fucose, the substrate of fucosyl transferases. Several cell-surface antigens, including the leukocyte Lewis system and cell-surface antigens in pathogenic bacteria, depend on the availability of GDP-l-fucose for their expression. Therefore, the enzyme is a potential target for therapy in pathological states depending on selectin-mediated cell-to-cell interactions. Previous crystallographic investigations have shown that GDP-4-keto-6-deoxy-d-mannose epimerase/reductase belongs to the short-chain dehydrogenase/reductase protein homology family. The enzyme active-site region is at the interface of an N-terminal NADPH-binding domain and a C-terminal domain, held to bind the substrate. The design, expression and functional characterization of seven site-specific mutant forms of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase are reported here. In parallel, the crystal structures of the native holoenzyme and of three mutants (Ser107Ala, Tyr136Glu and Lys140Arg) have been investigated and refined at 1. 45-1.60 A resolution, based on synchrotron data (R-factors range between 12.6 % and 13.9 %). The refined protein models show that besides the active-site residues Ser107, Tyr136 and Lys140, whose mutations impair the overall enzymatic activity and may affect the coenzyme binding mode, side-chains capable of proton exchange, located around the expected substrate (GDP-4-keto-6-deoxy-d-mannose) binding pocket, are selectively required during the epimerization and reduction steps. Among these, Cys109 and His179 may play a primary role in proton exchange between the enzyme and the epimerization catalytic intermediates. Finally, the additional role of mutated active-site residues involved in substrate recognition and in enzyme stability has been analyzed.

Amino Acid Substitution↗
PubMed2000

Leukocyte adhesion deficiency II-from A to almost Z.

Leukocyte adhesion deficiency (LAD) type II is the second human disorder identified which involves the adhesion cascade. While in LAD I the integrin family is defective, in LAD II the selectin system is involved. The syndrome has been described in only five patients and is transmitted as an autosomal recessive trait. The infectious episodes and the severity are much milder than those observed in LAD I, and the only persistent clinical symptom is chronic severe periodontitis. Delay separation of the umbilical cord, which is a hallmark for LAD I, was not observed in any of the LAD II patients. The exact defect in the system is absence of the SLeX, which is an important ligand for the selectin on the leukocyte lead ing to a profound defect in leukocyte rolling, the first step in the adhesion cascade. This causes a marked decrease in chemotaxis accompanied by pronounced neutrophilia. Apart from the leukocyte defect, these patients suffer from severe growth and mental retardation and exhibit the rare Bombay blood group type. The primary defect in the syndrome is in fucose metabolism, with the absence of all fucosylated glycans on cell surface membranes. Recently, it is was found that the defect is in a specific transporter of GDP fucose into the Golgi apparatus, and thus no fucosylation process takes place, and no surface expression can be detected. The exact genetic defect in the transporter is still unknown. Four of the patients were of Arabic origin while the fifth was of Turkish origin. It seems that the primary defect is somewhat different and, therefore, fucose administration was effective in the Turkish child, but did not show any beneficial results in the patients of Arabic origin.

Cell Adhesion↗
PubMed1999

Structural and enzymatic characterization of human recombinant GDP-D-mannose-4,6-dehydratase.

GDP-D-mannose-4,6-dehydratase (GMD) is the key enzyme in the 'de novo' pathway of GDP-L-fucose biosynthesis. The reported cDNA sequences for human GMD predict three forms of different length, whose 'in vivo' occurrence and molecular properties are completely undefined. Here, we report the expression in Escherichia coli and the properties of each native recombinant GMD form. Only the 42 kDa long GMD (L-GMD) and the 40.2 kDa (M-GMD) forms were recovered as soluble functional proteins, while the 38.7 kDa form, short GMD (S-GMD), lacking an N-terminal domain critical for dinucleotide binding, was inactive and formed a precipitate. Both L-GMD and M-GMD are homodimers and contain 1 mol of tightly bound NADP+. Their kinetic properties (Km, Kcat) are apparently identical and both forms are non-competitively feedback-inhibited by GDP-L-fucose to a similar extent. M-GMD is the predominant enzyme form expressed in several human cell lines. These data seem to suggest that modulation of the 'de novo' pathway of GDP-L-fucose biosynthesis involves mechanisms other than differential 'in vivo' expression of GMD forms.

Amino Acid Sequence↗
PubMed1998

GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily.

BACKGROUND: The process of guanosine 5'-diphosphate L-fucose (GDP-L-fucose) biosynthesis is conserved throughout evolution from prokaryotes to man. In animals, GDP-L-fucose is the substrate of fucosyltransferases that participate in the biosynthesis and remodeling of glycoconjugates, including ABH blood group and Lewis-system antigens. The 'de novo' pathway of GDP-L-fucose biosynthesis from GDP-D-mannose involves a GDP-D-mannose 4,6 dehydratase (GMD) and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER). Neither of the catalytic mechanisms nor the three-dimensional structures of the two enzymes has been elucidated yet. The severe leukocyte adhesion deficiency (LAD) type II genetic syndrome is known to result from deficiencies in this de novo pathway. RESULTS: The crystal structures of apo- and holo-GMER have been determined at 2.1 A and 2.2 A resolution, respectively. Each subunit of the homodimeric (2 x 34 kDa) enzyme is composed of two domains. The N-terminal domain, a six-stranded Rossmann fold, binds NADP+; the C-terminal domain (about 100 residues) displays an alpha/beta topology. NADP+ interacts with residues Arg12 and Arg36 at the adenylic ribose phosphate; moreover, a protein loop based on the Gly-X-X-Gly-X-X-Gly motif (where X is any amino acid) stabilizes binding of the coenzyme diphosphate bridge. The nicotinamide and the connected ribose ring are located close to residues Ser107, Tyr136 and Lys140, the putative GMER active-site center. CONCLUSIONS: The GMER fold is reminiscent of that observed for UDP-galactose epimerase (UGE) from Escherichia coli. Consideration of the enzyme fold and of its main structural features allows assignment of GMER to the reductase-epimerase-dehydrogenase (RED) enzyme homology superfamily, to which short-chain dehydrogenase/reductases (SDRs) also belong. The location of the NADP+ nicotinamide ring at an interdomain cleft is compatible with substrate binding in the C-terminal domain.

Amino Acid Sequence↗
PubMed1998

Preliminary crystallographic investigations of recombinant GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from E. coli.

The GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GM_ER) isolated from E. coli has been overexpressed as a GST-fusion protein and purified to homogeneity. The enzyme, an NADP+(H)-binding homodimer of 70 kDa, is responsible for the production of GDP-L-fucose. GM_ER shows significant structural homology to the human erythrocyte protein FX, which is involved in blood-group glycoconjugate biosynthesis, displaying 3,5 epimerase/reductase activity on GDP-4-keto-6-deoxy-D-mannose. GM_ER has been crystallized in a trigonal crystalline form, containing one molecule per asymmetric unit, suitable for high-resolution crystallographic investigations.

Amino Acid Sequence↗
PubMed1998

Defective intracellular activity of GDP-D-mannose-4,6-dehydratase in leukocyte adhesion deficiency type II syndrome.

Leukocyte adhesion deficiency type II (LAD II) is a rare genetic disease characterized by severe immunodeficiency which is related to defective expression in leukocytes of sialyl-Lewis X (SLeX), a fucosylated ligand for endothelial selectins. The molecular basis of LAD II is still unknown, but has been tentatively localized in the de novo pathway of GDP-L-fucose biosynthesis from GDP-D-mannose. Here, we demonstrate that in cell lysates from a LAD II patient, GDP-D-mannose-4,6-dehydratase (GMD), the first of the two enzymes of the pathway has a defective activity compared to control subjects. GMD in cell lysates from both parents showed intermediate activity levels. Cloning of GMD from patient and control lymphocytes ruled out any mutation affecting the amino acid GMD sequence and the purified recombinant proteins from both controls and the patient showed identical specific activities. Since the levels of immunoreactive GMD in cell lysates were comparable in the patient and in controls, the biochemical deficiency of intracellular GMD activity in LAD II seems to be due to mutation(s) affecting some still unidentified GMD-regulating protein.

Cloning, Molecular↗
PubMed1998

Synthesis and characterization of a specific peptide nucleic acid that inhibits expression of inducible NO synthase.

Inducible nitric oxide synthase (iNOS) is modulated at the transcriptional level. Overexpression of this protein may result in high levels of nitric oxide leading to tissue damage and immunosuppression. In order to reduce the pathological effects of NO overproduction many efforts have been devoted to the identification of specific inhibitors of iNOS. The discovery of peptide nucleic acids (PNA), a novel class of molecules able to selectively interact with nucleic acids, prompted us to attempt a new way for the regulation of NO production. Here we describe the synthesis, characterization and in vitro effects of a PNA molecule bearing a homopyrimidine sequence complementary to the 5' coding region of murine iNOS mRNA. This PNA shows specific interactions with iNOS mRNA in RNase protection assays and is able to block the synthesis of iNOS protein selectively in a rabbit reticulocyte lysate system. These results strengthen the view of a possible pharmacological application of PNA as a compound able to interfere with a specific enzymatic activity even at low concentrations.

Animals↗